Christopher Apostolatos

Inhibition of atypical protein kinase C‑ι effectively reduces the malignancy of prostate cancer cells by downregulating the NF-κB signaling cascade

Prostate cancer (PC) is the most common type of cancer among men. Aggressive and metastatic PC results in life- threatening tumors, and represents one of the leading causes of mortality in men. Previous studies of atypical protein kinase C isoforms (aPKCs) have highlighted its role in the survival of cultured prostate cells via the nuclear factor (NF)-κB pathway. The present study showed that PKC-ι was overexpressed in PC samples collected from cancer patients but not in non-invasive prostate tissues, indicating PKC-ι as a possible prognostic biomarker for the progression of prostate carcinogenesis. Immunohistochemical staining further confirmed the association between PKC-ι and the prostate malignancy. The DU-145 and PC-3 PC cell lines, and the non-neoplastic RWPE-1 prostatic epithelial cell line were cultured and treated with aPKC inhibitors 2-acetyl-1,3-cyclopentanedione (ACPD) and 5-amino-1-(1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1). Western blot data demonstrated that ICA-1 was an effective and specific inhibitor of PKC-ι and that ACPD inhibited PKC-ι and PKC-ζ. Furthermore, the two inhibitors significantly decreased malignant cell proliferation and induced apoptosis. The inhibitors showed no significant cytotoxicity towards the RWPE-1 cells, but exhibited cytostatic effects on the DU-145 and PC-3 cells prior to inducing apoptosis. The inhibition of aPKCs significantly reduced the translocation of NF-κB to the nucleus. Furthermore, this inhibition promoted apoptosis, reduced signaling for cell survival, and reduced the proliferation of PC cells, whereas the normal prostate epithelial cells were relatively unaffected. Overall, the results suggested that PKC-ι and PKC-ζ are essential for the progression of PC, and that ACPD and ICA-1 can be effectively used as potential inhibitors in targeted therapy.

Oncogenic PKC-ι activates Vimentin during epithelial-mesenchymal transition in melanoma; a study based on PKC-ι and PKC-ζ specific inhibitors

ABSTRACT Melanoma is one of the fastest growing cancers in the United States and is accompanied with a poor prognosis owing to tumors being resistant to most therapies. Atypical protein kinase Cs (aPKC) are involved in malignancy in many cancers. We previously reported that aPKCs play a key role in melanomas cell motility by regulating cell signaling pathways which induce epithelial-mesenchymal Transition (EMT). We tested three novel inhibitors; [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1T) along with its nucleoside analog 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) which are specific to protein kinase C-iota (PKC-ι) and 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) which is specific to PKC-zeta (PKC-ζ) on cell proliferation, apoptosis, migration and invasion of two malignant melanoma cell lines compared to normal melanocytes. Molecular modeling was used to identify potential binding sites for the inhibitors and to predict selectivity. Kinase assay showed >50% inhibition for specified targets beyond 5 μM for all inhibitors. Both ICA-1 and ζ-Stat significantly reduced cell proliferation and induced apoptosis, while ICA-1 also significantly reduced migration and melanoma cell invasion. PKC-ι stimulated EMT via TGFβ/Par6/RhoA pathway and activated Vimentin by phosphorylation at S39. Both ICA-1 and ζ-Stat downregulate TNF-α induced NF-κB translocation to the nucleus there by inducing apoptosis. Results suggest that PKC-ι is involved in melanoma malignancy than PKC-ζ. Inhibitors proved to be effective under in-vitro conditions and need to be tested in-vivo for the validity as effective therapeutics. Overall, results show that aPKCs are essential for melanoma progression and metastasis and that they could be used as effective therapeutic targets for malignant melanoma.

Abstract 3934: Analysis of PKC-ζ protein and mRNA levels in normal and malignant breast tissue

It is estimated that in 2015 breast cancer will be the second leading cause of cancer death in women. For this purpose, biochemical markers for breast cancer have been investigated, to assist in early detection and more accurate diagnoses. In breast cancer tissue, the atypical isozyme of protein kinase C zeta, PKC-ζ, has been a topic in research; It has been shown that an overexpression of this protein may be indicative of developing carcinogenesis and contributes to proliferation through the NF kappa B pathway, which is a stress-regulated switch for cell survival. In this investigation, the expression of PKC-ζ was analyzed in normal and malignant female human breast tissue samples by Western blot, immunoprecipitation and PCR. In the preliminary results, the malignant breast tissue samples illustrated a significant expression of PKC ζ when compared to the expression of PKC-ζ in normal breast tissue samples. The same tissues were also processed for total RNA isolation which was followed by cDNA synthesis and Real Time PCR. The level of PKC-ζ mRNA was tested and no overexpression was observed in either malignant or normal breast tissue samples. While protein studies may suggest that PKC-ζ could be considered a biomarker for breast cancer, the same cannot be said about mRNA levels. The overexpression of PKC-ζ protein level and the normal PKC-ζ mRNA level are indicators of possible miRNA activity that may regulate translation in malignant tissues but not the normal. Citation Format: Christopher Apostolatos, Tracess Smalley, Mildred A. Duncan. Analysis of PKC-ζ protein and mRNA levels in normal and malignant breast tissue. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3934.

Abstract POSTER-THER-1428: Effects of atypical PKC inhibitors on ovarian cancer proliferation and RNA levels

Ovarian cancers are highly lethal tumors which account for approximately four percent of all women’s cancers and are the fifth leading cause of cancer-related death among women. At diagnosis the majority of patients have metastatic disease and the long-term survival remains low. Certain ovarian cancers are highly lethal tumors due to the emergence of therapy-resistant ovarian cancer cells. Protein kinase C-iota (PKC-ι) has been shown to aid in the ability of cancer cells to resist drug-induced apoptosis. Recently, it has been reported that PKC-ι which is located in chromosome 3 at 3q26.2 is the most common genomic amplicon as identified by comparative genomic hybridization [Proc Natl Acad Sci U S A 102:12519–12524(2005)]. Additionally, an increase in PKC-ι DNA copy number was associated with decreased progression-free survival of ovarian cancer patients. Moreover, only PKC-ι gene amplification is highly correlated with protein overexpression, tumor size, lymph node metastasis and clinical stage out of four genes studied on the 3q26 amplification [Genes Chromosomes Cancer 47:127-136(2008)]. The focus of this research was to determine the in-vitro efficacy of the pan-atypical PKC inhibitor, 2-acetyl-1,3-cyclopentanedione (ACPD; Diabetes. 2014 Apr 4) and the novel PKC-iota inhibitor, ICA-1, (1H-imidazole-4-carboxamide, 5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy) methyl]cyclopentyl]-,[1R-(1α, 2β, 3 β, 4 α) [The Inter. J. Biochem. & Cell Biol. 43:784-794(2011)] on HEY ovarian cancer cell proliferation and RNA concentration. In contrast to ACPD which inhibits both PKC-ι and PKC-zeta (PKC-ζ), ICA-1 specifically inhibits the activity of PKC-ι but not (PKC-ζ). The effects of ACPD and ICA-1on HEY proliferation was quantified by Trypan blue exclusion assay. RNA was isolated from treated and untreated cells using the Rneasy Mini Kit and protocol. Total RNA concentration was estimated using the NanoDrop1000 Spectrophotometer. Results showed that incubation of HEY ovarian cancer cells with ICA-1(1 μm) decreased proliferation by 41% compared to controls at 72 hours post-treatment. ICA-1(2 μM) reduced RNA levels by 17% at 72 hours post-treatment compared to controls. In contrast, ACPD (2 μM) inhibited proliferation by 82% compared to controls and RNA levels were reduced by 97% at 24 hours post-treatment. These results suggest the potential of ICA-1 and ACPD as chemotherapeutic agents. Citation Format: Minjel Shah, Christopher Apostolatos, Hercules Apostolatos, Mildred Acevedo-Duncan. Effects of atypical PKC inhibitors on ovarian cancer proliferation and RNA levels [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1428.

Abstract 1037: Inverse regulation of p53 by atypical PKC inhibitors in ovarian cancer cells

Ovarian cancers are highly lethal tumors which account for approximately four percent of all women9s cancers and are the fifth leading cause of cancer-related death among women. At diagnosis, the majority of patients have metastatic disease and the long-term survival remains low. Certain ovarian cancers are highly lethal tumors due to the emergence of therapy-resistant ovarian cancer cells. Protein kinase C-iota (PKC-ι) has been shown to aid in the ability of cancer cells to resist drug-induced apoptosis. Recently, it has been reported that PKC-ι, which is located in chromosome 3 at 3q26.2, is the most common genomic amplicon as identified by comparative genomic hybridization [Proc Natl Acad Sci U S A 102:12519-12524(2005)]. Additionally, an increase in PKC-ι DNA copy number was associated with decreased progression-free survival of ovarian cancer patients. Moreover, only PKC-ι gene amplification is highly correlated with protein overexpression, tumor size, lymph node metastasis and clinical stage out of four genes studied on the 3q26 amplification [Genes Chromosomes Cancer 47:127-136(2008)]. The focus of this research was to determine the regulation of p53 by pan-atypical PKC inhibitor, 2-acetyl-1,3-cyclopentanedione (ACPD) [Diabetes. 63:2690-2701 (2014)] and the novel PKC-ι inhibitor, ICA-1, (1H-imidazole-4-carboxamide, 5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy) methyl]cyclopentyl]-,[1R-(1α, 2β, 3 β, 4 α) [The Inter. J. Biochem. & Cell Biol. 43:784-794(2011)]. In contrast to ACPD which inhibits both PKC-ι and PKC-zeta (PKC-z), ICA-1 specifically inhibits the activity of PKC-ι. The effects of ACPD and ICA-1 on HEY (serous surface epithelial tumor cells) and T80 (non-malignant ovarian cells) proliferation was quantified by Trypan blue exclusion assay. Results showed that incubation of HEY ovarian cancer cells with ICA-1 (1 μm) decreased proliferation by 12% compared to controls at 72 hours post-treatment. In contrast, ACPD (2 μM) inhibited proliferation by 81% compared to controls at 24 hours post-treatment. Incubation of T80 cells with ICA-1 resulted in increased proliferation, which is the opposite effect seen in the malignant cells. Through Western blot analysis, we found that HEY cells treated with 2μM ICA had lower levels of phosphorylated p53 compared to the control while T80 cells treated with 2μM ICA-1 had higher levels of phosphorylated p53 compared to the control. This pattern was also seen with phosphorylated PKC-ι. We propose that the mechanism involves inverse regulation of p53 by atypical PKC inhibitor ICA-1 in the normal ovarian cancer cells versus the malignant cells. Normally, MDM2 negatively regulates and degrades p53, but this phenomenon is reversed in the non-malignant cells after treatment with ICA-1. These results suggest that PKC-ι is involved in the regulation of p53. Citation Format: Minjel Shah, Christopher Apostolatos, Hercules Apostolatos, Mildred Acevedo-Duncan. Inverse regulation of p53 by atypical PKC inhibitors in ovarian cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1037. doi:10.1158/1538-7445.AM2015-1037